Bf066 Antiplatelet And Antithrombotic Effect And Its Mechanism

Background and objective:Arterial thrombotic diseases, such as heart attack and stroke, are the leading cause of morbidity and mortality worldwide. Platelet activation plays essential role in the initiation and development of these atherothrombotic diseases. Accordingly, Antiplatelet therapy has been established as a cornerstone in the management of thrombosis and vascular diseases.Some P2Y12receptor antagonists have been reported to affect platelet function through additional mechanisms. In this study, we exploredthe effect of BF066in the process of thrombosis formation.Methods:①Aggregation of human washed platelets in response to agonist or antagonists was analyzed using lumi-aggregometer;②platelet secretion was monitored by measuring ATP release using CHRONO-LUME reagent on Chrono-Log lumi-aggregometer in parallel with aggregation;③the effects of BF066on TXA2production was measured by ELISA;④the assay for cAMP-PDE activity was performed using HPLC;⑤platelet spreading assays were performed by Leica DM5500Q microscope;⑥intravital microscopy ofFeCl3-induced thrombosis in mouse mesenteric arteriolewasmeasured by Leica DM5500Q microscope;⑦aggregation of mice platelets in response to agonist or antagonists was analyzed using lumi-aggregometer;⑧to test the possible bleeding side effect of BF066, we measured BF066-induced C57BL/6mouse blood loss after tail snip at the same concentration (29mg/kg, a single bolus i.v. injection) used for in vivo antithrombotic study.Results:①BF066inhibits platelet aggregation and ATP releasing induced by multiple platelet agonists in a dose-dependent manner.②Platelets are activated via a complex signal transduction cascade mediated by multiple agonists including ADP, thrombin, collagen and thromboxane. In the range of10-100μM, BF066also abolished or drastically inhibited platelet aggregation and ATP release in human washed platelets induced by arachidonicacid, collagen, U46619, AYPGKF and thrombin.③BF066did not affect platelet TXA2production.④The aggregation inhibition induced by ADP is potentiated by adenosine and can be dramatically antagonized by the A2A antagonist SCH58261.⑤The inhibition of BF066on PDE activity was concentration dependent.⑥In the range of3-30μM, BF066inhibited platelet spreading on immobilized fibrinogen in a dose-dependent manner.⑦BF066was effective to inhibit thrombus formation in vivo.⑧The blood loss induced by BF066is97±39μl, which is similar to the vehicle (72±57μl, P>0.05), and negligible compared to the blood loss of459±282μl induced by clopidogrel (30mg/kg, p.o., once daily for two days).Conclusion:Taken together, these results suggest that BF066may be an effective and safe antiplatelet agent targeting both PDE and A2A.Considering the successful use of combined antiplatelet therapy, BF066may be further developed as a noveldual target antiplatelet agent.